The present invention relates to peptides immunoreactive with antibodies to Toxoplasma gondii (to be referred to as T. gondii hereinafter), nucleic acid sequences encoding these peptides, recombinant vector molecules comprising these nucleic acid sequences, host cells transformed with the recombinant vector molecule, immunochemical reagents comprising the peptides or antibodies directed against the peptides, a test kit for the detection of T. gondii infections as well as a vaccine for the protection against T. gondii infections.
T. gondii is an intracellular protozoan parasite found throughout the world and capable of infecting all species of mammals and all types of cells within a given individual. T. gondii is classified as a coccidian with two life cycles, asexual and sexual.
In the asexual stage, T. gondii exists in different forms, for instance; tachyzoites, pseudocysts, bradyzoites, and oocysts. The tachyzoite is the obligate intracellular form of T. gondii which characterizes acute infection. The infective stage of T. gondii for human host cells is the pseudocyst. The pseudocyst has a diameter of 30-100 micrometers and contains hundreds to thousands of infectious units termed bradyzoites. Infection is frequently initiated by ingestion of pseudocysts present in raw or uncooked unfrozen meats. In addition, infection can occur by ingestion of oocysts in the faeces of cats experiencing active intestinal infection. Infections may also be acquired through blood or leukocyte transfusion, by organ transplantation or by transplacental transmission during pregnancy (Wilson et al., J. Exp. Med. 151, 328-346, 1980). The wall of either the pseudocyst or the oocyst is broken down in the small intestine by host digestive enzymes releasing the bradyzoites or sporozoites, respectively, which then penetrate the columnar epithelium. It is probable that bradyzoites or sporozoites reach the liver by the hematogenous route where they are ingested by Kxc3xcpffer cells. Once inside a cell, the organisms are referred to as tachyzoites. Liver parenchymal cells also become infected (Kranenbxc3xchl and Remington, Immun. of Parasitic Infections; eds. Cohen, S., Warren, K. S. London: Blackwell Scientific Publications pp. 356-421, 1982; Remington and Kranenbxc3xchl, Immun. of Human Infection, part II, Edited by Nahmias, A. J., O""Reilly, R. J. New York, Plenum Medical Book Company, pp. 327-371, 1982).
Replication immediately follows entry into host cells. In the host, macrophages transport bradyzoites throughout the body. The bradyzoites survive and replicate within the macrophage parasitophorous vacuole by preventing the fusion of lysosomes with it. Replication results in the lysis of the host cell. Organisms are phagocytosed by new macrophages or other cell types and repeat the cycle.
The sexual stage of T. gondii occurs only in feline hosts (Miller et al., J. Parasitology, 58, p. 928-937, 1972). The intermediate host (e.g., mouse) becomes infected by ingesting either oocysts or pseudocysts. The mouse develops pseudocysts throughout its own tissues. The cat becomes infected when it eats infected meat (e.g., rodent tissues) containing the pseudocysts or ingests oocysts. Bradyzoites or sporozoites penetrate columnar epithelial cells and differentiate into merozoites. Following replication, merozoites rupture infected epithelial cells and infect adjacent ones. Some merozoites differentiate into pre-sex cells termed macrogametocytes (female form) and microgametocytes (male form). The microgametocytes fuse with macrogametocytes, forming zygotes termed oocysts (Dubey and Frenkel, J. Protozool. 19:155-177, 1972). Oocysts enter lumen of the small intestine and are defecated. Each oocyst sporulates,in the soil, producing eight infectious sporozoites, the infectious stage for the intermediate host.
The bradyzoite is the form that encysts approximately 8-10 days after acquisition in vivo and characterizes the chronic, latent phase of infection. Thus, the bradyzoite form is the only stage that has the ability to initiate the enteroepithelial cycle.
T. gondii induces a mild or unapparent disease in healthy adults but causes a severe disease, toxoplasmosis, or even death in congenitally infected children and in immunocompromised patients. Primary infection of pregnant women occurs in European countries with frequencies between 0.2 and 1.0%. In approximately 40-50% of the cases, the unborn child is infected. Infection in the fetus during pregnancy will (in approximately 10% of the cases) lead to neonatal death or a severely multi- handicapped child, but in 90% of the cases the child will be born with an asymptomatic, latent infection (Desmonts and Couvreur, Ann. Pediatr. 1984, 31, 805-809; Alford et al. Bull. NY Acad. Med. 1974, 50, 160-181). Up to 85% of the patients with latent congenital toxoplasmosis will develop significant sequelae including one or more episodes of active retinochoroiditis. Other clinical symptoms are inflammation, lymphadenitis, encephalitis and fever.
In immunocompromised patients, especially in the case of AIDS, T. gondii causes a severe pathology. In approximately 30 percent of Toxoplasma-antibody positive patients with AIDS, toxoplasma encephalitis will develop due to reactivation of their latent infection. In immunocompetent humans, T. gondii infection induces a long-lasting protective immunity against reinfection (Remington and Krahenbxc3xchl, 1982)
There are several strains of T. gondii known. One of the most important strains is the RH-strain which is highly virulent and originally isolated from human brain tissue.
Most investigations in the past regarding T. gondii were focused on the identification and molecular characterization of tachyzoite-specific antigens. More than 1000 different T. gondii-specific proteins have been identified. Surface proteins of T. gondii have been studied extensively. Of these surface-proteins the RH strain p30 surface protein is the most abundant; it constitutes approximately 5% of the total tachyzoite protein. The p30 surface protein is recognized intensively by human IgM, IgG, IgA and IgE antibodies (Decoster et al., Clin. Exp. Immunol., 73, 376-382, 1988; Godard et al., Infect. Immun., 58, 2446-2450, 1990) and is therefore useful for diagnostic purposes. Using monoclonal antibodies directed against p30, two immunocapture tests have been developed for the detection of anti-Toxoplasma IgM (Cesbron et al., J. Immunol. Methods, 83, 151-158, 1985) and IgA (Decoster et al., Lancet, ii, 1104-1106, 1988) antibodies.
The present invention provides new peptides, immunoreactive with antibodies to T. gondii, that can be used in diagnosing T. gondii infections in humans.
In particular the present invention provides peptides, immunoreactive with antibodies to Toxoplasma gondii, comprising part of the amino acid sequences as shown in SEQ ID No.: 1.
A preferred embodiment of the present invention are peptides comprising at least part of the amino acid sequences shown in SEQ ID No. 3 or 5.
Another preferred embodiment of the present invention are polypeptides comprising polymeric forms of said peptides.
The term xe2x80x9cpeptidexe2x80x9d as used herein refers to a molecular chain of amino acids with a biological activity, and does not refer to a specific length of the product. Thus inter alia, proteins, oligopeptides, polypeptides and fusion-peptides as well as fusion-proteins are included.
The term xe2x80x9cpolypeptidexe2x80x9d refers to dimeric, trimeric, . . . , polymeric forms of any length of a peptide according to the present invention.
The term xe2x80x9cpeptidexe2x80x9d as used herein further refers to so-called functional variants, for example, acid addition salts, amides, esters, and specifically C-terminal esters, and N-acyl derivatives of the peptides according to the invention. These functional variants are considered to be part of the present invention. Also included are peptides which are modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation. It will be understood that for the particular proteins or polypeptides embraced herein, natural variations can also exist. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions from which can be expected that they do not essentially alter biological and immunological activities, have been described. Amino acid replacements or conservative replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atla of protein sequences and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3).
The term xe2x80x9cpart of the amino acid sequencexe2x80x9d as used herein means an amino acid sequence comprising a subsequence of a peptide of the invention. Said parts or fragments are peptides comprising at least one antigenic determinant of the amino acid sequence as shown in SEQ ID No.: 1. Examples of these fragments are for instance peptides comprising the amino acid sequence as shown in SEQ ID No.: 3 or 5. Fragments can inter alia be produced by enzymatic cleavage of precursor molecules, using restriction endonucleases for the DNA and proteases for the polypeptides. Other methods include chemical synthesis of the fragments or the expression of peptide fragments by DNA fragments.
Suitable antigenic fragments of a peptide according to the invention containing (an) epitope(s) can be found by means of the method described in Patent Application WO 86/06487, (Geysen, H. M. et al. (Proc. Natl. Acad. Sci. 81, 3998-4002, 1984;, Geysen, H. M. et al. J. Immunol. Meth. 102, 259-274, 1987) based on the so-called pepscan method, wherein a series of partially overlapping peptides corresponding with partial sequences of the complete polypeptide under consideration, are synthesized and their reactivity with antibodies is investigated.
The minimal antigenic fragments of the epitopes can be found by synthesizing a series of partially overlapping peptides consisting of a gradually increase in amino acids per scan. The minimal immunoreactive core-structure of the epitope can be used to synthesize polymeric synthetic peptides on branched lysine molecules. Application of these branched peptides in a diagnostic assay results in an increase in
The peptides comprising part of the amino acid sequence as shown in SEQ ID No.: 1 (representing clone #114) and preferred the peptides according to the present invention comprising the amino acid sequence as shown in SEQ ID No.: 3 or 5, are recognized by human anti-T. gondii antibodies of the IgG- as well as IgM-class respectively, and are therefore suitable diagnostic markers for T. gondii infections.
In SEQ ID No.: 1 the amino acid sequence are given for proteins with a (calculated) molecular weight of approximately 25.6 kD.
The preparation of the peptides or fragments thereof according to the invention is effected by means of one of the known organic chemical methods for peptide synthesis or with the aid of recombinant DNA techniques. This latter method involves the preparation of the desired peptide by means of bringing to expression a recombinant polynucleotide with a polynucleotide sequence which is coding for one or more of the peptides in question in a suitable micro-organism as host.
The organic chemical methods for peptide synthesis are considered to include the coupling of the required amino acids by means of condensation reaction, either in homogeneous phase or with the aid of a so-called solid phase.
The condensation reaction can be carried out as follows:
a) condensation of a compound (amino acid, peptide) with a free carboxyl group and protected other reactive groups with a compound (amino acid, peptide) with a free amino group and protected other reactive groups, in the presence of a condensation agent;
b) condensation of a compound (amino acid, peptide) with an activated carboxyl group and free or protected other reaction groups with a compound (amino acid, peptide) with a free amino group and free or protected other reactive groups.
Activation of the carboxyl group can take place, inter alia, by converting the carboxyl group to an acid halide, azide, anhydride, imidazolide or an activated ester, such as the N-hydroxy-succinimide, N-hydroxy-benzotriazole or p-nitrophenyl ester.
The most common methods for the above condensation reactions are: the carbodiimide method, the azide method, the mixed anhydride method and the method using activated esters, such as described in The Peptides, Analysis, Synthesis, Biology Vol. 1-3 (Ed. Gross, E. and Meienhofer, J.) 1979, 1980, 1981 (Academic Press, Inc.).
Preparation of suitable fragments of above-mentioned peptides according to the invention using the xe2x80x9csolid Phasexe2x80x9d is for instance described in J. Amer. Chem. Soc. 85, 2149 (1963) and Int. J. Peptide Protein Res. 35, 161-214 (1990).
As already indicated above, the peptides according to the invention can likewise be prepared with the aid of recombinant DNA techniques. For example, the peptides according to the invention can be incorporated in a repeating sequence (xe2x80x9cin tandemxe2x80x9d) or can be prepared as a constituent of a (much larger) protein or polypeptide. This type of peptides therefore likewise falls within the scope of the invention.
For this purpose, as a constituent of a recombinant DNA, a nucleic acid sequence is used which codes for a peptide according to the invention and which, furthermore, is substantially free from nucleic acid segments, which in the naturally occurring T. gondii genome flank the nucleic acid sequence indicated above.
This latter method involves the preparation of the desired peptide by means of bringing to expression a recombinant polynucleotide with a nucleic acid sequence which is coding for one or more of the peptides in question in a suitable micro organism as host.
The invention further encompasses nucleic acid sequences encoding the peptides according to the present invention and nucleic acid sequences comprising part of the nucleic acid sequence shown in SEQ ID No.: 2. Fragments of these nucleic acid sequences as shown in SEQ ID No.: 2 are for instance the nucleic acid sequences as shown in SEQ ID No.: 4 or 6. A further object of the present invention are nucleic acid sequences encoding peptides as shown in SEQ ID No.: 3 or 5.
The invention also comprises (a) host cell(s) transformed or transfected with a nucleic acid sequence or recombinant expression vector molecule, capable of producing the peptides according to the invention by expression of the corresponding nucleic acid sequence.
xe2x80x9cNucleic acid sequencexe2x80x9d as used herein refers to a polymeric form of nucleotides of any length, both to ribonucleic acid sequences and to deoxy ribonucleic acid sequences. In principle, this term refers to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, as well as double and single stranded RNA, and modifications thereof.
A nucleic acid sequence according to the present invention can be ligated to various replication effecting DNA sequences with which it is not associated or linked in nature resulting in a so called recombinant vector molecule which can be used for the transformation of or transfection into a suitable host. Useful recombinant vector molecules, are preferably derived from, for example plasmids, bacteriophages, cosmids or viruses.
Specific vectors or cloning vehicles which can be used to clone nucleic acid sequences according to the invention are known in the art and include inter alia plasmid vectors such as pBR322, the various pUC, pGEM and Bluescript plasmids, bacteriophages, e.g. lambda gt-Wes, Charon 28 and the M13 derived phages or viral vectors such as SV40, adenovirus, Semliki Forest Virus, vaccinia virus, Herpes viruses or polyoma virus (see also Rodriquez, R. L. and D. T. Denhardt, ed., Vectors: A survey of molecular cloning vectors and their uses, Butterworths, 1988; Lenstra, J. A. et al., Arch. Virol. 110, 1-24, 1990). The methods to be used for the construction of a recombinant vector molecule according to the invention are known to those of ordinarily skill in the art and are inter alia set forth in Maniatis, T. et al. (Molecular Cloning A Laboratory Manual, second edition; Cold Spring Harbor Laboratory, 1989).
For example, the insertion of the nucleic acid sequence according to the invention into a cloning vector can easily be achieved when both the genes and the desired cloning vehicle have been cut with or either one of them has been digested with the same restriction enzyme(s) as complementary DNA termini are thereby produced.
The recombinant vector molecules may additionally contain one or more marker activities that may be used to select for desired transformants, such as ampicillin and tetracycline resistance in pBR322, as for example ampicillin resistance and xcex1-peptide of xcex2-galactosidase in pUC8.
A suitable host cell is a micro-organism or cell which can be transformed by a nucleic acid sequence encoding a polypeptide or by recombinant vector molecule comprising such a nucleic acid sequence and which can if desired be used to express said polypeptide encoded by said nucleic acid sequence. The host cell can be of procaryotic origin, e.g. bacteria such as Escherichia coli, Bacillus subtilis and Pseudomonas species; or of eucaryotic origin such as yeasts, e.g. Saccaromyces cerevisiae or higher eucaryotic cells such as insect, plant or mammalian cells, including HeLa cells and Chinese hamster ovary (CHO) cells.
Information with respect to the cloning and expression of the nucleic acid sequence of the present invention in eucaryotic cloning systems can be found in Esser, K. et al. (Plasmids of Eucaryotes, Springer-Verlag, 1986).
In general, prokaryotes are preferred for the construction of the recombinant vector molecules useful in the invention. For expression nucleic acid sequences of the present invention are introduced into an expression vector, i.e. said sequences are operably linked to expression control sequences. Such control sequences may comprise promoters, enhancers, operators, inducers, ribosome binding sites etc. Therefore, the present invention provides a recombinant vector molecule comprising a nucleic acid sequence encoding the peptides identified above operably linked to expression control sequences, capable of expressing the DNA sequences contained therein in (a) transformed host cell(s).
It should, of course, be understood that the nucleotide sequences inserted at the selected site of the cloning vector may include only a fragment of the complete nucleic acid sequence encoding the peptides according to the invention as long as the transformed or transfected host will produce a polypeptide having at least one or more antigenic determinants.
In order to purify the expressed polypeptides produced as described above, host cells transformed with a recombinant vector according to the invention are cultured in an adequate volume and the polypeptides produced are isolated from such cells or from the medium if the protein is excreted. Polypeptides excreted into the medium can be isolated and purified by standard techniques, e.g. salt fractionation, centrifugation, ultrafiltration, chromatography, gel filtration or immunoaffinity chromatography, whereas intra cellular polypeptides can be isolated by first collecting said cells, disrupting the cells, for example by sonication or by other mechanically disruptive means such as French press followed by separation of the polypeptides from the other intracellular components and forming isolated polypeptides. Cell disruption could also be accomplished by chemical (e.g. EDTA or detergents such as Triton X114) or enzymatic means such as lysozyme digestion.
Antibodies, immunoreactive with a peptide according to the invention are also part of the present invention.
The peptides or fragments thereof prepared and described above are used to produce antibodies, both polyclonal and monoclonal. Monoclonal antibodies directed against peptides according to the invention are considered to be part of the present invention.
The preparation of cell lines producing monoclonal antibodies may occur by, for example, the Kxc3x6hler and Milstein technique (Kxc3x6hler and Milstein devised the techniques that resulted in the formation monoclonal antibody-producing hybridomas (G. Kxc3x6hler and C. Milstein, 1975, Nature 256:495; 1976, Eur. J. Immunol. 6:511-519)), transformation technique of B-lymphocytes with a fusion partner being either a human or a mouse-human hybrid myeloma cell line, or a direct fusion of an EBV-tranformed B cell line with said myeloma cell lines.
An immunochemical reagent comprising one or more peptides or antibodies according to the invention is also part of the present invention.
The term xe2x80x9cimmunochemical reagentxe2x80x9d according to the invention usually consists of one more peptides according to the invention or antibodies according to the invention and a suitable support or a labeling substance. Supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an aldehyde particle (such as a ceramic magnetizable particle with active aldehyde surface groups), an erythrocyte, a dye sol, a metal sol or metal compound as sol particle, a carrier protein such as BSA or KLH.
Labeling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, a metal sol or metal compound as sol particle.
In a method for the detection of antibodies directed against T. gondii in a sample, an immunochemical reagent according to the invention is brought into contact with the sample. After which, the presence of immune complexes formed between the peptide and antibodies in the sample is detected and by this detection the presence of T. gondii antibodies in the sample is known and can be determined quantitatively.
Depending on the nature and further characteristics of the immunochemical reagent the immunochemical reaction that takes place is a so called sandwich reaction, an agglutination reaction, a competition reaction or an inhibition reaction.
For the detection of T. gondii in a sample an immunochemical reagent according to the invention, containing one or more peptides according to the invention, can be brought into contact with the sample and anti-T. gondii antibodies after which the presence of immune complexes formed can be detected and, from this, the presence of T. gondii in a sample can be determined.
A particularly suitable method for the detection of T. gondii in a sample is based on a competition reaction between a peptide according to the invention provided with a labeling substance and a T. gondii antigen (present in the sample) whereby the peptide and the antigen are competing with the antibody directed against T. gondii attached to a solid support.
An antibody according to the invention may also be brought into contact with a sample after which the presence of immune complexes formed is detected which is a measure for the presence of T. gondii in the sample.
A test kit according to the invention may comprise as an essential constituent an immunochemical reagent as described above. For carrying out a sandwich reaction, for the detection of T. gondii antibodies the test kit may comprise, for example, the peptide according to the invention coated directly or via a carrier-protein, i.e. BSA, to a solid support, for example the inner wall of a microtest well, and either a labeled peptide according to the invention or a labeled anti-antibody.
For carrying out a competition reaction, the test kit may comprise a peptide according to the invention coated to a solid support, and a labeled antibody directed against T. gondii preferably a monoclonal antibody directed against said peptide.
In an agglutination reaction the test kit comprises an immunochemical reagent which may comprise a peptide according to the invention coated to particles or sols. Another embodiment of a test kit is, for example, the use of a labeled peptide according to the invention as immunochemical reagent in a competition reaction with a T. gondii antigen to be detected for a binding site on the antibody directed against T. gondii, which is coated to a solid support.
It is within the scope of this invention to use the new nucleic acid sequences according to the invention as the basis of a test to detect T. gondii by a nucleic acid amplification technique for instance the polymerase chain reaction (PCR) or the nucleic acid sequence based amplification (NASBA), as described in EP 201,814 and EP 329,822, respectively. A method for the amplification and the detection of a T. gondii nucleic acid sequence in a sample using at least one nucleic acid sequence or fragment thereof according to the invention primer(s) in order to perform a nucleic acid amplification of said T. gondii nucleic ac id sequence and to detect the amplified sequence is also part of the present invention. Part of the invention is also a test amplification technique, said kit containing at least a set of primers corresponding to at least a part of the nucleotide sequences according to the invention.
It i s within the scope of this invention to use the new peptides or fragment s thereof according to the invention as t he basis of transport of co-expressed heterologeous proteins to the surface of the plasma membrane of T. gondii or another parasitic organism or eucaryotic host cell via a phosphatidylinositol glycan (PI-G) moiety. A method for the intracellular transport and surface expression of a heterologeous polypeptide or fragments thereof by using at least the hydrophobic NH2-terminal signal peptide sequence that might be cleaved by an NH2-terminal signal peptidase and the hydrophobic COOH-terminal peptide that in some way interacts with a putative transamidase that cleaves the peptide and concomitantly adds the PI-G-moiety. Part of the invention is also to use the NH2-terminal signal peptide and COOH-terminal peptide sequences for the development of a heterologeous expression cassettes in parasites and eucaryotic host cells.
Vaccines for the protection against Toxoplasma gondii are also part of the present invention. These vaccines comprise a host cell according to the invention or a peptide according to the invention or a polypeptide according to the invention, together with a pharmaceutical acceptable carrier.
The vaccine according to the invention can be administered in a conventional active immunization scheme: single or repeated administration in a manner compatible with the dosage formulation and in such amount as will be prophylactically effective, i.e. the amount of immunizing antigen or recombinant micro-organism capable of expressing said antigen that will induce immunity against challenge by virulent Toxoplasma gondii parasites. Immunity is defined as the induction of a significant level of protection in a population after vaccination compared to an unvaccinated group.
The administration of the vaccine can be d one, e.g. intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, orally or intranasally.
Additionally the vaccine may also contain an aqueous medium or a water containing suspension, often mixed with other constituents, e.g. in order to increase the activity and/or shelf life. These constituents may be salts, pH buffers, stabilizers (such as skimmed milk or casein hydrolysate), emulsifiers, adjuvants to improve the immune response (e.g. oils, muramyl dipeptide, aluminiumhydroxide, saponin, polyanions and amphipatic substances) and preservatives.